Lithium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Accessibility of sulfhydryl residues induced by cytochalasin B binding and conformational dynamics in the human erythrocyte glucose transporter

Studies with intact cells have implicated essential sulfhydryl groups in the carrier-mediated glucose transport of human erythrocytes. In an attempt to identify and characterize such essential sulfhydryl residues we have studied the interaction of p-chloromercuribenzoate (PCMB) with a purified glucose transporter preparation (band 4.5) from human erythrocytes, in the presence and absence of its ligands, and the effects of this interaction on the binding of cytochalasin B (CB) to the transporter. At least 3 mol of PCMB reacted per mol of this preparation. A portion of the reaction was significantly enhanced in the presence of cytochalasin B. This enhancement was a saturable function of CB concentration, and was half-maximal at a CB concentration equal to the dissociation constant for the CB binding to the preparation. This CB-sensitive, PCMB reaction product comigrated with the band 4.5 on lithium dodecyl sulfate-polyacrylamide gel electrophoresis. An excess of d-glucose did not affect the PCMB reaction by itself in the absence of CB, but totally abolished the CB-induced enhancement of the PCMB reaction. PCMB inhibited the CB binding activity of the transporter preparation, and this inhibition was also enhanced in the presence of CB. These results suggest that CB binding perturbs the conformational dynamics of the glucose transporter resulting in an exposure of at least two sulfhydryl residues to PCMB reaction, and that some of these CB-sensitive sulfhydryl groups are essential for CB binding to the transporter.

Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development

Changes in the amounts of thylakoid galactolipids and proteins were monitored for 96 hours following iron resupply to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) plants. During this period of iron nutrition-mediated chloroplast development, the amount of galactolipid per leaf area increased linearly with time. Assuming galactolipids are an index for the amount of thylakoids, then there was a linear synthesis of thylakoid membranes during regreening. Total thylakoid protein synthesis, however, lagged behind galactolipid synthesis, suggesting that proteins are inserted secondarily into the galactolipid matrix of the thylakoid membrane during development.Iron deficiency caused an increase in the free chlorophyll band under the conditions of gel electrophoresis used. Of the chlorophyll proteins resolved, the chlorophyll protein associated with photosystem I was most diminished in iron-deficient tissue, and appeared to recover most rapidly. Changes in the light-harvesting chlorophyll proteins are also discussed.The number of polypeptides resolved by lithium dodecyl sulfate-polyacrylamide gel electrophoresis was higher in iron-deficient thylakoids. During regreening, the number of resolved polypeptides decreased.

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at −196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ƒ, cytochrome b-559 and cytochrome b 6, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at −196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.

Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans

Cultures of the cyanobacterium Anacystis nidulans were grown under iron-deficient conditions and then restored by the addition of iron. Membrane proteins from iron-deficient and iron-restored cells were analyzed by lithium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The incorporation of [(35)S]sulfate into membrane proteins and lactoperoxidase-catalyzed (125)I iodination were used to monitor the rates of polypeptide biosynthesis and surface exposure of membrane proteins, respectively. These polypeptide profiles revealed major differences in the membrane composition of iron-deficient and normal cells. Iron deficiency caused a decrease in the amount of certain important membrane proteins, reflecting a decreased rate of biosynthesis of these peptides. Several photosystem II peptides also showed an increase in surface exposure after iron stress. In addition, iron deficiency led to the synthesis of proteins at 34 and 52 kilodaltons which were not present in normal cells. When iron was restored to a deficient culture, a metabolic sequence was initiated within the first 12 h after the addition of iron which led to phenotypically normal cells. Pulse labeling with [(35)S]sulfate during this period demonstrated that iron addition initiates a coordinated pattern of synthesis that leads to the assembly of normal membranes.

Chemical nature of protein complex of photoreaction unit including reaction center in chromatophores of photosynthetic bacterium, Rhodospirillum rubrum, as detected by successive dissociation method.

Reaction center of chromatophores of Rhodospirillum rubrum consists of three kinds of protein, H-, M-, and L-subunit, and is bound with many other kinds of protein to form a larger protein complex (PRU; photoreaction unit), which contains all the bacteriochlorophyll. In the present study, purified PRU was dissociated in a stepwise manner in the presence of various mixtures of lithium dodecyl sulfate, sodium cholate and/or sodium deoxycholate, and separated into five, smaller protein complexes (PL1, PL2, PL3, PL4, and PL4') by high-speed molecular-sieve chromatography. The protein complexes were analyzed for molecular mass (Mm), protein composition, and molecular weights of the constituent proteins by the chromatography described above and by lithium or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggest that PRU consisted of 1 molecule each of 40K, 39K, 31K (H-subunit), 25K (M-subunit), and 22K (L-subunit), about 12 molecules each of 12K (light-harvesting bacteriochlorophyll-protein) and 11K, and about 6 molecules each of 10K and 9K (the protein nomenclature refers to the apparent molecular weights); the measured and calculated Mm values were 650K and 547K, respectively. The compositions of the other protein complexes were as follows. PL1 = PRU-10K-9K (measured & calculated Mm, 520K & 409K); PL2 = PL1-39K (340K & 267K); PL3 = PL2-40K (160K & 147K); PL4 = PL3-31K-25K (90K & 82K); PL4' = 31K + 25K + 22K (inactivated reaction center) (90K & 78K). The molar ratios of 12K and 11K to 25K were lower in the dissociated protein complexes than in PRU, and they differed from one complex to another. The locations of the constituent proteins in PRU are discussed.

Biosynthesis and Membrane Insertion of a Chlorophyll a/b-binding Protein by Isolated Vicia faba L., Chloroplasts

Summary Chloroplasts isolated from leaves of Vicia faba L. were capable of incorporating [35S]methionine in the light into a number of thylakoid-bound polypeptides. Among these products, two polypeptides co-migrated in lithium dodecylsulfate polyacrylamide gel electrophoresis with the constituting polypeptides of the light-harvesting chlorophyll a/b-protein complex II. After labelling of chloroplasts in vitro, chlorophyll-protein complex II was precipitated with a monospecific antiserum as revealed by gel electrophoresis. The immunoprecipitate was shown to be radioactive by fluorography. One-dimensional peptide mapping of the products of hydrolysis with S. aureus protease confirmed that the radioactivity was an integral part of the apoprotein of the chlorophyll-protein complex II. Incorporation of [35S]methionine into this complex by isolated chloroplasts was inhibited by D-threo-chloramphenicol but not by cycloheximide. Labelling did not occur if the chloroplasts were isolated from leaves which had been pretreated with α-amanitin. It is concluded that in intact isolated Vicia faba chloroplasts chlorophyll-protein complex II is synthesized from mRNA of nuclear origin.

Characterization of New Strains of Nonphotosynthetic Mutants of <italic>Chlamydomonas reinhardtii</italic> III. Photosystem II-Related Thylakoid Proteins in Five Mutants and Double Mutants

PS II-enriched particles of the wild type, of three mutants and of two double mutants of Chlamydomonas reinhardtii were analyzed by lithium dodecylsulfate polyacrylamide gel electrophoresis at 4°C. The mutant Pg 27 was devoid of light-harvesting Chl-protein complex (CP) CP II, but had normal cytochrome b-559 and displayed all wild type photochemical activities. The mutant Fl 50 lacked a pool of cytochrome b-559 photooxidizable at 77 K but was able to photooxidize a second pool at 293 K in the presence of FCCP; it showed some weak PS II activity. The mutant Fl 39 lacked both these cytochrome b-559 pools and did not display any PS II activity. The double mutants Fl 39 Pg 28 and Fl 50 Pg 27 had defects similar to those of their respective parents Fl 39 or Fl 50 but, in addition, they were devoid of Chi b and of CP II. In these four mutants having impaired PS II function, five proteins of Mr=50,000, 47,000, 33,000, 27,000 and 19,000 were totally (Fl 39, Fl 39 Pg 28) or partly (Fl 50, Fl 50 Pg 27) missing. The first two of these proteins corresponded to the apoproteins of CP III and IV. These results pointed out a strong correlation between these five proteins, cytochrome b-559 and PS II primary photochemistry. In mutation and cross experiments, these five PS II-associated proteins and cytochrome b-559 appeared to be linked characters controlled by nuclear gene(s), but they behaved independently of CP II.

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca 2+ + Mg 2+)-ATPase activity of about 10 nmol (min·mg) −1 and an ATP-dependent Ca 2+ uptake of about 10 nmol (min·mg) −1. When incubated in the presence of Mg[γ- 32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [ 32P]phosphate-labeled Ca 2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca 2+ or Ca 2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ( M r = 94 000, 87 000, 60 000 and 43 000 ). Some minor calmodulin-binding proteins were enriched in the membrane fractions ( M r = 69 000, 57 000, 39 000 and 37 000 ). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca 2+ uptake is essentially unaltered, while the Ca 2+ capacity is diminished as a consequence of a doubling in the rate of Ca 2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca 2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.

Biosynthesis and Assembly of Thylakoid Membrane Proteins in Isolated Chloroplasts from Vicia faba L.: The P700-Chlorophyll a-Protein

Summary Isolated Vicia faba chloroplasts were incubated with [ 35 S]methionine in the light, and the newly formed polypeptide products associated with thylakoid membranes were separated by lithium dodecylsulfate polyacrylamide gel electrophoresis (LiDS-PAGE). At least 30 different labeled polypeptides could be distinguished on fluorographs of the gels. Part of them did not comigrate with any stained bands of proteins, but a number of about 15 corresponded in size to preexisting thylakoid components. The inhibition pattern obtained with inhibitors of photophosphorylation (CCCP, DCMU) and translation (cycloheximide, D-threo-chloramphenicol, ribonuclease) indicates that labeling of all these polypeptides occurred in intact chloroplasts only and was energized by photophosphorylation. One of the labeled products comigrated with P700-chlorophyll a-protein (CP I). This labeled protein was precipitated specifically by direct immunoprecipitation and in two-dimensional crossed immunoelectrophoresis using monospecific antibodies against purified CP I apoprotein. Moreover, it was indistinguishable from CP I by the number and size of peptides formed by digestion with S. aureus protease. On this basis, it is suggested that CP I apoprotein was synthesized and assembled by isolated chloroplasts. Polypeptides labeled in isolated chloroplasts comigrated with the α and β subunits of chloroplast coupling factor CF 1 . The patterns of the radioactive fragments obtained from these labeled polypeptides by limited proteolysis corresponded precisely to the patterns of stainable fragments derived from subunits of the purified enzyme. A 32 kd polypeptide was also identified by one-dimensional peptide mapping as a translation product of isolated Vicia faba chloroplasts.

Isolation and development of chlorosomes in the green bacterium Chloroflexus aurantiacus.

Freeze-fracture electron microscopy was used to study further the changes in chlorosome structure during the development of the photosynthetic apparatus in Chloroflexus aurantiacus J-10-fl. During development, in response to decreased light intensity or lower oxygen tension, the number of chlorosomes per cell increased. The same conditions also led to a general thickening of chlorosomes but did not affect their length or width. The thickening of the chlorosomes paralleled increases in the bacteriochlorophyll c/bacteriochlorophyll a ratio. Semiaerobic induction of the photosynthetic apparatus did not produce a synchronous assembly of chlorosomes in all cells of a given culture. Even adjacent cells of a single filament showed great variations in the rate and extent of response. Parallel appearance of (i) approximately 5-nm particles (in a lattice configuration) in the membrane attachment site, (ii) the crystalline baseplate material (with a periodicity of approximately 6 nm) adjacent to the membrane attachment site, and (iii) the chlorosome envelope layer preceded addition of longitudinally oriented, rodlike elements (diameter, congruent to 6 m) to the chlorosome core. It is estimated that each chlorosome can funnel energy into approximately 100 reaction centers. Chlorosomes could be isolated by a simple density gradient procedure only from cells grown at low light intensity. A bacteriochlorophyll a species absorbing at 790 nm was associated with isolated chlorosomes. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorosomes showed only a few low-molecular-weight polypeptides (less than 15,000).